Compositions and methods for the maintenance of oral health

ABSTRACT

The invention provides compositions comprising one or more isolated LDH-deficient  mutans streptococcus  strains and one or more isolated  S. oralis  strains and/or one or more isolated  S. uberis  strains. Compositions of the invention are useful to maintain oral health, by for example treating and/or preventing one or more symptoms of dental caries, periodontitis and/or other oral cavity diseases or wounds.

PRIORITY

This application claims the benefit of U.S. Provisional patentapplication Ser. No. 60/494,169, filed Aug. 11, 2003.

BACKGROUND OF THE INVENTION

Dental caries is characterized by dissolution of the mineral portion ofthe tooth, which can result in pain and loss of viability of the tooth,necessitating costly repair or extraction of the tooth. Dental cariesaffects 50% of children aged 5-9 years, 67% of adolescents age 12-17years, and 94% of adults aged >18 years in the US (Morbidity andMortality Weekly Reports 51: 144-147, 2002). Clean teeth will not decay;however, even with vigorous cleaning it is difficult to keep teethsufficiently clean. Various methods have been developed to prevent oralleviate dental caries including, for example, the addition of sodiumfluoride, sodium silicofluoride or hydrofluosilicic acid to drinkingwater, and sodium fluoride or tin fluoride to topical preparations,including dentifrices and mouthrinses. The prevention of caries bycoating teeth with polymeric materials or sealants has been used;however, these techniques are costly, can require etching of the teethwith phosphoric acid and can be effective only in young children whohave not yet developed caries. Antibacterial agents, includingantibiotics, have also been proposed as a treatment for dental caries.Antibiotics kill microorganisms that are responsible for producing acidin the mouth such as Streptococcus mutans, but antibiotics are notselective in the killing of oral bacteria and also kill beneficialbacteria present in the oral cavity. This can result in a microbialimbalance in the mouth, which can have serious consequences. Therefore,more effective methods for the treatment and prevention of dental cariesare needed in the art.

Actinobacillus actinomycetemcomitans (Aa) is the principal etiologicagent of early-onset periodontitis including localized and generalizedprepubertal periodontitis, localized and generalized juvenileperiodontitis, and rapidly progressive or refractory adultperiodontitis. Tooth loss is the ultimate detrimental effect ofdestructive periodontal disease. A national survey of the United Statesrevealed a prevalence of localized juvenile periodontitis of 0.53% andof generalized juvenile periodontitis of 0.13%. Loe & Brown, J.Periodontol. 62:608-616 (1991). Findings from a number of studiescorroborate the conclusion that early-onset disease is similar in otherindustrialized countries and is more frequent in developing countries.Loe & Brown, J. Periodontol. 62:608-616 (1991).

In addition, certain types of adult periodontitis, which in general arevery common conditions affecting over half the adult population, arelikely to be caused by a select group of microorganisms indigenous tothe oral cavity. These include Aa, Porphyronzonas gingivalis, Prevotellaintermedia, Bacteroides forsythus, Treponema denticola, Campylobacterrectus and Eikenella corrodens. There are antibiotic, surgical, andmechanical therapies for the treatment of the various types ofperiodontitis, but no means for prevention. Tetracycline has been widelyused in the treatment of early-onset periodontitis. There remains aconcern, however, of strains developing resistance to tetracycline aswell as the possibility of overgrowth of other pathogenicmicroorganisms. Given the incidence of periodontal diseases, safepreventative and treatment strategies are needed in the art. Control ofperiodontal disease is also very important in light of recent attentionto the possible role of periodontal infections as risk factors forsystemic disease (e.g., coronary heart disease). Therefore, methods oftreatment and prevention of early-onset periodontitis, localized andgeneralized juvenile periodontitis, and rapidly progressive orrefractory adult periodontitis are needed in the art.

BRIEF SUMMARY OF THE INVENTION

The instant invention provides methods and compositions for themaintenance of oral health, such as the treatment and/or prevention ofperiodontitis, dental caries, Candida or fungal overgrowth in an oralcavity, halitosis, xerostomia-induced dental caries, oral bacterialinfections or diseases, and oral wounds. In one embodiment the inventionprovides probiotics for the maintenance of oral health.

Probiotics are viable single or mixed culture microorganisms, which whenapplied to animals or humans, beneficially affect their host byimproving the properties of the indigenous microflora. Traditionally,probiotic use has focused on the general category of gastrointestinalhealth, but the approach of using beneficial organisms has beensuggested to prevent or treat other conditions, including application tomaintain vaginal and urinary tract health. In the instant inventionprobiotics are used to maintain oral health.

One embodiment of the invention provides a composition comprising one ormore isolated Streptococcus oralis strains and/or one or more S. uberisstrains combined with one or more isolated mutans streptococcus strainsthat are lactate dehydrogenase-deficient. The mutans streptococcusstrains can be one or more LDH-deficient strains of S. rattus, S.cricetus, S. mutans, S. sobrinus, S. downeii, S. macacae, and S. ferus.The composition can further comprise a carrier. The mutans streptococcusstrain can be a naturally-occurring strain or a genetically modifiedstrain that is lactate dehydrogenase-deficient. A mutans streptococcusstrain can be, for example; a S. rattus strain JH145. A S. oralis straincan be, for example, S. oralis strain KJ3 or KJ3sm. A S. uberis straincan be, for example, KJ2 or KJ2 sm.

Another embodiment of the invention provides a food compositioncomprising one or more isolated S. oralis strains and/or one or moreisolated S. uberis strains, and one or more isolated mutansstreptococcus strains, wherein the mutans streptococcus strains arelactate dehydrogenase-deficient.

Still another embodiment of the invention provides a dentifrice, chewinggum, lozenge, oral rinse, or topical agent composition comprising one ormore isolated S. oralis strains and/or one or more isolated S. uberisstrains, and one or more isolated mutans streptococcus strains, whereinthe mutans streptococcus strains are lactate dehydrogenase-deficient.

Yet another embodiment of the invention provides a method formaintaining oral health of a subject comprising administering to an oralcavity of a subject a composition comprising one or more isolated S.oralis strains and/or one or more isolated S. uberis strains and one ormore isolated mutans streptococcus strains, wherein the mutansstreptococcus strains are lactate dehydrogenase-deficient. Thecomposition can be administered to the subject about once a day, aboutonce a week or about once a month. The subject can be a mammal, such asa human. Maintaining oral health can comprise the treatment, prevention,or both treatment and prevention of periodontitis, dental caries,Candida or fungal overgrowth, halitosis, xerostomia-induced dentalcaries or periodontitis, oral bacterial infections or diseases, oralwounds or a combination thereof.

Even another embodiment of the invention provides a method ofnon-persistently colonizing an oral cavity of a subject withtherapeutically-effective bacteria comprising administering to the oralcavity of a subject a combination comprising one or more isolated S.oralis strains and/or one or more isolated S. uberis strains, and one ormore isolated mutans streptococcus strains, wherein the mutansstreptococcus strains are lactate dehydrogenase-deficient. The subjectcan be a mammal, such as a human.

Therefore, the invention provides methods and compositions for themaintenance of oral health, including, for example, the preventionand/or treatment of dental caries, periodontitis, Candida or fungalovergrowth, halitosis, or xerostomia-induced dental caries orperiodontal disease oral bacterial infections or diseases, oral woundsor a combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of administration of daily treatment withJH145.

FIGS. 2A-C show the comparison of a S. mutans JH145 ldh gene to a S.mutans BHT-2 ldhgene and the corresponding protein sequences.

DETAILED DESCRIPTION OF TIE INVENTION

Probiotics can be defined as the administration of live microorganismsin adequate amounts to confer a health benefit on the host. Thoughoriginally developed for “gut health”, probiotics are now beinginvestigated in immune system modulation, vaginal and urinary tracthealth, allergies, inflammatory disorders and hypertension. Bacteria arenormal inhabitants of humans, and the oral cavity provides an ecologicalniche for over 300 microbial species. Microbial interactions arelogically of enormous importance in controlling the ecology of dentalplaque and, thus, the outcome: oral health or disease. One preventiveapproach is to encourage colonization and growth of protective speciesor the establishment of a microbial flora that is balanced in favor ofhealth. Beneficial effects can involve the production of a specificenzyme(s) or metabolite(s), or the probiotic organism can also cause thebody to produce the beneficial action. A beneficial effect can also beachieved by inhibition of colonization or outgrowth of a pathogenicmicroorganism by competition for nutrients or attachment sites.

The invention provides compositions, therapeutic systems and methods ofuse for the maintenance of oral health including, for example, thetreatment and/or prevention of dental caries, periodontitis, oralbacterial infections and diseases, oral wounds, Candida or fungalovergrowth, halitosis, or xerostomia-induced dental caries orperiodontal disease, the promotion of wound healing, or a combinationthereof in a subject. A composition of the invention comprises atherapeutically effective amount of one or more isolated strains ofLDH-deficient mutans streptococcus in combination with a therapeuticallyeffective amount of one or more isolated strains of S. oralis and/or oneor more isolated strains of S. uberis.

Streptococcus oralis and Streptococcus uberis

Streptococcus oralis (previously known as S. sanguis Type II) and S.uberis are important components in maintaining the normal, healthybalance of microorganisms that compose the periodontal flora. See,Socransky et al., Oral Microbiol. Immunol. 3:1-7 (1988); Hillman andShivers, Arch. Oral. Biol., 33:395-401 (1988); Hillman, et al., Arch.Oral. Biol., 30:791-795 (1985). S. oralis produces hydrogen peroxide,which can inhibit periodontal pathogens such as Actinobacillusactinomycetemcomitans (Aa), Bacteroides forsythus, and P. intermedia.Therefore, S. oralis and S. uberis can be useful in the maintenance oforal health. Compositions of the invention comprise one or more isolatedstrains of S. oralis, for example, ATCC 35037, ATCC 55229, ATCC 700233,ATCC 700234 and ATCC 9811. Other strains of S. oralis include KJ3 andKJ3sm. KJ3sm is a naturally occurring genetic variant of KJ3 that isresistant to streptomycin. The streptomycin resistance is advantageousbecause it provides a marker for easy isolation of the bacteria.Additionally, streptomycin resistant strains are slightly attenuated anddo not survive as long in an oral cavity as wild-type strains. Thisproperty is useful where the goal is to non-persistently colonize theoral cavity of an animal with the bacteria.

S. uberis in plaque has been found to correlate with periodontal health,in particular by interfering with the colonization by periodontalpathogens such as Porphyromonas gingivalis, Campylocbacter recta, andEikenella corrodens. Compositions of the invention can comprise one ormore isolated strains of S. uberis, for example, ATCC 13386, ATCC 13387,ATCC 19435, ATCC 27958, ATCC 35648, ATCC 700407, ATCC 9927, strain KJ2or strain KJ2sm. KJ2sm is a naturally occurring genetic variant of KJ2.That is streptomycin resistant and provides the same advantages as forstreptomycin-resistant strains of S. oralis. One or more isolatedstrains of S. oralis or one or more isolated strains of S. uberis, orboth, can be used in compositions and methods of the invention.

Mutans Streptococcus

Compositions of the invention comprise one or more isolated mutansstreptococcus bacteria species deficient in the production of lacticacid. These strains include, for example, S. rattus, S. cricetus, S.mutans, S. sobrinus, S. downeii, S. macacae, and S. ferus. A mutansstreptococcus strain of the invention does not substantially produceL(+) lactate dehydrogenase (LDH). Such a strain is termed anLDH-deficient strain. An LDH-deficient strain of mutans streptococcusproduces 75%, 80%, 90%, 95%, 98%, 99%, or 100% less lactic acid thanwild-type strains of mutants streptococcus. An LDH-deficient mutansstreptococcus strain can be a naturally occurring strain of mutansstreptococcus or a genetically modified strain of mutans streptococcus.LDH-deficient mutans streptococcus can compete with and/or displacepathogenic bacteria such as S. mutans, a principal etiological agent ofdental caries, in the oral cavity. LDH-deficient mutans streptococcusstains will compete with S. mutans for the same nutrients, colonizationsites, etc. in an oral cavity when administered as a probiotic.Therefore, LDH-deficient mutans streptococcus strains can be used to,for example, prevent and/or treat dental caries. LDH-deficient strainsof mutans streptococcus are non-pathogenic, alter the microenvironmentof the oral cavity to prevent colonization or outgrowth of pathogenicorganisms, and/or displace pathogenic organisms from the oral cavitywhere the pathogen is part of the host's indigenous flora.

Examples of LDH-deficient mutans streptococcus strains include, forexample, S. rattus JH145 (ATCC 31377) (a spontaneous,naturally-occurring LDH-deficient mutant) (See FIGS. 2A-C for a JH145mutant LDH nucleotide and polypeptide sequence) and JH140 (ATCC 31341)(a chemically-modified LDH-deficient mutant). See e.g., Stanshenko &Hillman, Microflora of plaque in rats following infection with anLDH-deficient mutant of Streptococcus rattus, Caries Res. 23:375-377(1989); Hillman, Lactate dehydrogenase mutants of Streptococcus mutans:Isolation and preliminary characterization. Infect. Immun. 21:206-212(1978); see also Abhyankar et al., Serotype c Streptococcus mutansmutatable to lactate dehydrogenase deficiency. J Dent Res November 1985;64(11):1267-71.

An LDH-deficient strain of mutans streptococcus can be derived from amutans streptococcus strain using, for example, chemical or physicalmutagenesis techniques. Strains that are mutagenized using thesetechniques are considered genetically modified strains. For example, amutans streptococcus strain can be subjected to mutagens such as nitrousacid, formic acid, sodium bisulphate, LV light, base analog mutagens,including for example, 5-bromo-deoxyuridine (5BU), alkylators such asethyl methane sulfonate (EMS), methyl methane sulfonate (MMS),diethylsulfate (DES), and nitrosoguanidine (NTG, NG, MNNG). See e.g., InVitro Mutagenesis Protocols, Braman, Ed., Humana Press, 2002.

Naturally-occurring, spontaneous LDH-deficient mutans streptococcusstrains can be prepared using methods disclosed in, for example,Hillman, Lactate dehydrogenase mutants of Streptococcus mutans:isolation and preliminary characterization. Infect. Immun. 21:206-212(1978). Spontaneous LDH-deficient mutants occur at the rate ofapproximately 10⁻⁵ frequency. See Johnson et al., Cariogenic potentialin vitro in man and in vivo in the rat of lactate dehydrogenase mutantsof Streptococcus mutans. Arch. Oral Biol. 25:707-713 (1980).

Naturally-occurring, spontaneous LDH-deficient strains of mutansstreptococcus can be differentiated from LDH-producing strains of mutansstreptococcus by plating the bacteria on glucose tetrazolium medium.LDH-deficient mutans streptococcus colonies will be bright red andrelatively larger in size than colonies of the parent strain, which arewhite and relatively smaller in size on the glucose tetrazolium medium.Naturally-occurring, spontaneous LDH-deficient strains of mutansstreptococcus can be used in a composition of the invention.

An LDH-deficient strain of S. rattus has been isolated. Briefly, aculture of S. rattus BHT-2 was grown overnight to saturation in ToddHewitt broth, and diluted samples were spread on glucose tetrazoliummedium to give approximately 300 colonies per plate. Wild-type, acidproducing colonies are white on this medium. LDH-deficient mutants arebright red. S. rattus JH145 was one red colony amid approximately100,000 white colonies that were screened. S. rattus JH145 is thereforea naturally-occurring, LDH-deficient mutant.

LDH-deficient strains of mutans streptococcus, such as LDH-deficientmutants of S. rattus BHT-2, produce less total titratable acid whenincubated in the presence of glucose and other sugars or polyols, makesubstantially less lactic acid when incubated in the presence of glucosein the case of resting and growing cultures, adhere better tohydroxyapitite and accumulate more plaque when grown in the presence ofsucrose. LDH activity can be assayed as described by Brown &Wittenberger (J. Bacteriol. 110:604, 1972).

Terminal pH can be determined by subculturing strains (1:100) inTodd-Hewitt broth containing 1% glucose. After 48 hours incubation incandle jars at 37° C., the absorbance at 580 nm and pH of the culturescan be determined. Lactic acid concentration of cultures can bedetermined by gas-liquid chromatography. See Salanitro & Muirhead,Quantitative method for the gas chromatographic analysis of short-chainmonocarboxylic and dicarboxylic acids in fermentation media. Appl.Microbiol. 29:374-381 (1975); Hillman et al., Acetoin production bywild-type strains and a lactate dehydrogenase-deficient mutant ofStreptococcus mutans. Infect. Immun. 55:1399-1402 (1987).

Additionally, any genetic modification techniques known to those ofskill in the art can be used to create an LDH-deficient mutansstreptococcus strain from an LDH-producing mutans streptococcus parentstrain. For example, an LDH gene or a portion of an LDH gene can bedeleted or mutagenized, including, for example, insertional mutagenesistechniques. Other mutagenesis techniques include, for example,homologous recombination, recursive sequence recombination,oligonucleotide-directed mutagenesis, site-directed mutagenesis,error-prone PCR, phosphothioate-modified DNA mutagenesis,uracil-containing template mutagenesis, gapped duplex mutagenesis, pointmismatch repair mutagenesis, repair-deficient host strain mutagenesis,radiogenic mutagenesis, deletion mutagenesis, restriction-selectionmutagenesis, restriction-purification mutagenesis, site saturationmutagenesis, ensemble mutagenesis, recursive ensemble mutagenesis, andchimeric nucleic acid creation. Therefore, any genetic modificationtechnique that disables an LDH gene can be used to produce anLDH-deficient mutans streptococcus strain.

In one embodiment of the invention, the LDH-deficient strains, whethernaturally-occurring or genetically-modified mutants, have a reversionfrequency less than 10-7 and produce less than 10% of the parental levelof lactate dehydrogenase activity.

Compositions of the Invention

Compositions of the invention comprise one or more isolated strains ofmutants streptococcus combined with one or more isolated strains of S.oralis, or one or more isolated strains of S. uberis, or both one ormore isolated strains of S. oralis and one or more isolated strains ofS. uberis, wherein the mutans streptococcus strain is lactatedehydrogenase (LDH)-deficient. The combination of LDH-deficient mutansstreptococcus with S. oralis and/or S. uberis provides a significantpractical advantage in that the combination can used to prevent andtreat, for example, both dental caries and periodontitis. Treatment ofdental caries and/or periodontitis means that one or more symptoms ofdental caries and/or periodontitis is alleviated, reduced, prevented orameliorated either permanently or temporarily. Compositions and methodsof the invention can also be used to treat or prevent Candida or fungalovergrowth in an oral cavity, due to, for example, antibiotic treatment,to treat or prevent halitosis (bad breath), and to treat or preventdental caries and/or periodontitis associated with xerostomia (drymouth), to treat or prevent oral bacterial infections or diseases, totreat or prevent oral wounds and combinations thereof.

Mutans streptococcus, S. oralis and/or S. uberis strains of theinvention can be present in any therapeutically effective ratio.Therapeutically effective means effective to alleviate, reduce, preventand/or ameliorate one or more symptoms of dental caries, periodontitis,bacterial infections or diseases, oral wounds, Candida or fungalovergrowth, halitosis, or xerostomia-induced dental caries orperiodontal disease or a combination thereof either permanently ortemporarily. Therapeutically effective also means effective to promotewound healing in an oral cavity.

The bacterial strains of the invention can further comprise apharmaceutically acceptable or nutritionally acceptable carrier. Thecarrier is physiologically compatible with the oral cavity of thesubject to which it is administered. Carriers can be comprised ofsolid-based, dry materials for formulation into tablet, capsule,lozenge, or powdered form. A carrier can also be comprised of liquid orgel-based materials for formulations into liquid, gel, and chewing gumforms.

Suitable liquid or gel-based carriers include but are not limited to:water, physiological salt solutions, urea, alcohols and derivatives, andglycols (e.g., ethylene glycol or propylene glycol). Compositions of theinvention can also include natural or synthetic flavorings andfood-quality coloring agents, all of which are compatible withmaintaining viability of the bacterial strains of the invention.Thickening agents can also be added to compositions of the inventionsuch as corn starch, guar gum, carbopol, and xanthan gum.

Flavorings and/or colorants can also be included in the carrier.Compositions of the invention can also include a plasticizer such asglycerol or polyethylene glycol. The composition of the carrier can bevaried so long as it does not interfere significantly with thetherapeutic activity of the bacterial strains of the invention.

A composition can be formulated to be suitable for oral administrationin a variety of ways, for example in a liquid, a dried mass, adentifrice, a mouth wash, an oral rinse, a liquid suspension, a topicalagent, a powdered food supplement, a paste, a gel, a solid food, apackaged food, a wafer, lozenge, chewing gum and the like. Otherformulations will be readily apparent to one skilled in the art. Acomposition of the invention can include a nutrient supplement componentand can include any of a variety of nutritional agents, as are wellknown, including vitamins, minerals, essential and non-essential aminoacids, carbohydrates, lipids, foodstuffs, dietary supplements, and thelike.

Bacteria of the invention can be prepared in, for example, a fermenter.The bacteria can be harvested from the fermenter and can be, forexample, concentrated. Bacteria of the invention can be prepared for useby, for example, dehydration or spray drying. Spray drying generallycomprises spraying a suspension of bacteria in a vessel and under asteam of hot air. Bacteria can also be prepared for use bymicroencapsulation (see e.g., U.S. Pat. No. 6,251,478), freeze-drying,or by coating with a protective substance such as, for example, lipidmaterial such as triacylglycerols, waxes, organic esters, soybean oil,cottonseed oil, palm kernel oil, and esters of long-chain fatty acidsand alcohols.

Methods of Maintaining Oral Health

Pathogenic bacteria, such as S. mutans and Actinobacillusactinomycetemcomitans, which can colonize an oral cavity of an animal,can be inhibited and/or controlled by administering a compositioncomprising one or more LDH-deficient mutans streptococcus strains andone or more isolated S. oralis strains, or one or more isolated S.uberis strains, or both one or more isolated S. oralis strains and oneor more isolated S. uberis strains to an oral cavity. Compositions canbe administered to an oral cavity of a subject such as an animal,including a mammal, for example, a human, a non-human primate, a dog, acat, a rat, a mouse, a horse, a goat, or a rabbit. The bacterial strainsof the invention can form at least a part of the transient or indigenousflora of an oral cavity and exhibit beneficial prophylactic and/ortherapeutic effects in the cavity.

The invention provides methods for the treatment, prevention, or bothtreatment and prevention of dental carries, periodontitis, Candida orfungal overgrowth, halitosis, or xerostomia-induced dental caries orperiodontal disease oral bacterial infections or diseases, oral woundsor a combination thereof comprising administering a composition of theinvention to an oral cavity of a subject. Periodontitis includes, forexample, early-onset periodontitis, localized and generalized juvenileperiodontitis, and rapidly progressive or refractory adultperiodontitis. The composition is administered to the subject about oncea day, about once a week or about once a month.

The compositions of the invention can be orally administered in forexample, food, water, a dentifrice, a gel, a paste, an emulsion, aerosolspray, chewing gum, lozenge, tablet, capsule, or a liquid suspension.The bacteria can either be already formulated into food, water, gel orother carrier or can be a composition that is added to the carrier bythe user prior to consumption.

One embodiment of the invention provides a method of non-persistentlycolonizing an oral cavity of a subject with therapeutically-effectivebacteria comprising administering to the oral cavity of a subject acombination comprising one or more isolated strains of S. oralis and/orS. uberis and one or more isolated strains of mutans streptococcusorganisms, wherein the mutans streptococcus organisms are lactatedehydrogenase-deficient. In one embodiment of the invention theadministered bacterial strains do not permanently colonize the oralcavity, rather the strains are present in the oral cavity for about 1day, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2months or about 3 months after administration of the bacteria.Optionally, the bacteria can permanently and persistently colonize theoral cavity of a subject.

Compositions of the invention can be administered at a dose of about1×10³, 1×10⁵, 1×10⁷, 1×10⁹, or 1×10¹¹ CFU of viable bacteria. One, two,or more doses can be administered per day for about 1 week, about 2weeks, about 1 month, about 2 months, about 3 months, about a year ormore. Alternatively, a dose can be administered about every other day,about once a week, about once a month or about yearly.

EXAMPLE 1

Mutans streptococci for Maintenance of Oral Health

Daily treatment with JH145 results in decreased levels of indigenousStreptococcus mutans because of competition for nutrients, attachmentsites, and other factors. The reduced levels of indigenous S. mutanspromotes dental health, since this microorganism is closely associatedwith dental caries.

Seventy-two weanling ( 24 day-old) Sprague-Dawley rats were infectedwith S. mutans strain NG8 by pipetting 100 microliters of a suspensioncontaining approximately 10¹⁰ cells per ml into their mouths using amicropipettor. The infection regimen was repeated twice on consecutivedays. The animals were maintained on diet TD 80406 (Harlan Teklad) andwater ad lib, caged separately. NG8 was allowed to establish itself for4 weeks. The animals were randomly divided into 6 groups of 12 andtreated daily for 16 weeks as follows:

-   -   a. Animals in group 1 received 100 microliters of a suspension        of JH145 containing 10¹⁰ cells per ml using a micropipettor.    -   b. Animals in group 2 received 100 microliters of a suspension        of JH145 containing 10⁹ cells per ml using a micropipettor.    -   c. Animals in group 3 received 100 microliters of a suspension        of JH145 containing 10⁸ cells per ml using a micropipettor.    -   d. Animals in group 4 received 100 microliters of a suspension        of JH145 containing 10⁷ cells per ml using a micropipettor.    -   e. Animals in group 5 received 100 microliters of a suspension        of JH145 containing 10⁶ cells per ml using a micropipettor.    -   f. Animals in group 6 are sham treated.

At weekly intervals, plaque and saliva were sampled using cotton tippedswabs, and samples plated on S. mutans screening/selection medium toselectively cultivate NG8 (white colonies) and JH145 (red colonies).Levels of NG8 were plotted as a function of time. See FIG. 1.

Using a Univariate Analysis of Variance (ANOVA) to examine differencesamong the six groups (i.e., 5 treatment and 1 control) during week 9,results indicated a significant group effect, F (5, 63)=5.53, p<0.001.The effect size (ES=0.31) and observed power (Φ=0.99) for this analysisindicated that there was a sufficient amount of power to detect thissmall effect between the groups. Follow up t tests with Bonferronicorrection (p=0.01) were conducted using a priori planned comparisons,which tested each treatment group against the control. These analysesindicated that treatment groups 1-4 exhibited significantly less S.mutans NG8 expression relative to the control group, t's (1,21)≧2.7,p≦0.01.

Therefore, daily treatment of rats with >b 10 ⁶ cells of JH145 resultsin decreased levels of indigenous S. mutans. A decreased risk of dentalcaries and improved dental health should follow. The effect of thetreatment is likely to be significantly better in human subjects whowould hold and swish the probiotic in their mouths for 30 to 60 secondsper treatment. Relative to the rat, which quickly swallows the probioticpreparation, the longer exposure of human teeth to JH145 should increasethe opportunity for it to compete with indigenous S. mutans, resultingin improved effectiveness. Human studies can be performed to verify thatdaily exposure with a suspension of mutans streptococci for example, S.rattus JH145 (ldh,str) results in decreased numbers of an indigenous S.mutans strain and cessation of the exposure results in eventualelimination of S. rattus JH145 and return of the indigenous S. mutans tooriginal levels. Baseline levels of indigenous S. mutans can bedetermined. Daily doses of S. rattus JH145 can be administered to theoral cavity of the subjects. The oral flora of the subjects can besampled weekly and wild-type S. mutans and S. rattus JH145 levelsmonitored until a decrease in wild-type S. mutans levels is observed. S.rattus JH145 doses are discontinued and oral flora is sampled weekly tofollow decline in S. rattus JH145 levels and increase in wild-type S.mutans levels

Streptococcus oralis and Streptococcus uberis for Maintenance ofPeriodontal Health

Human studies can be done to verify that daily exposure with asuspension of S. oralis (str) and/or S. uberis results in decreasednumbers of indigenous periodontopathic species and cariogenic speciesand that cessation of the treatment results in eventual elimination ofthe S. oralis and/or S. uberis probiotic strain and return of theperiodontopathic species and cariogenic species to original levels.

For example, baseline levels of 4 periodontopathic bacteria in salivacan be determined for human subjects. Daily doses of S. oralis and/or S.uberis can be administered to the oral cavity of human subjects. Theoral flora of the human subjects can be sampled weekly to monitorpathogen and S. oralis and/or S. uberis levels until a decrease inlevels of one or more pathogens is observed. The S. oralis and/or S.uberis doses are stopped and oral flora of the human subjects is sampledweekly to follow the decline in S. oralis and/or S. uberis levels andthe corresponding increase in pathogen levels.

Finally, human studies and animal studies can be done to verify thatdaily exposure with a suspension of one or more isolated S. oralis (str)and/or S. uberis strains in combination with one or more isolatedLDH-deficient mutans streptococcus strains results in decreased numbersof indigenous periodontopathic species and that cessation of thetreatment results in eventual elimination of the probiotic strains andreturn of the periodontopathic species to original levels.

All patents, patent applications, and other scientific or technicalwritings referred to anywhere herein are incorporated by reference intheir entirety. The invention illustratively described herein suitablycan be practiced in the absence of any element or elements, limitationor limitations that are not specifically disclosed herein. Thus, forexample, in each instance herein any of the terms “comprising”,“consisting essentially of”, and “consisting of” may be replaced witheither of the other two terms. The terms and expressions which have beenemployed are used as terms of description and not of limitation, andthere is no intention that in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by embodiments, optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the description and theappended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

1. A composition comprising: (a) (i) one or more isolated Streptococcusoralis strains; (ii) or one or more isolated strains of Streptococcusuberis; (iii) or one or more isolated Streptococcus oralis strains andone or more isolated strains of Streptococcus uberis; and (b) one ormore isolated mutans streptococcus strains, wherein the mutansstreptococcus strains are lactate dehydrogenase-deficient.
 2. Thecomposition of claim 1, further comprising a carrier.
 3. The compositionof claim 1, wherein the mutans streptococcus strains are selected fromthe group consisting of S. rattus, S. cricetus, S. mutans, S. sobrinus,S. downeii, S. macacae, and S. ferus.
 4. The composition of claim 1,wherein the mutans streptococcus strain is S. rattus strain JH145. 5.The composition of claim 1, wherein the mutans streptococcus strain is anaturally-occurring mutant that is lactate dehydrogenase-deficient. 6.The composition of claim 1, wherein the S. oralis strain is S. oralisstrain KJ3sm or KJ3.
 7. The composition of claim 1, wherein the S.uberis strain is S. uberis strain KJ2sm or strain KJ2.
 8. Thecomposition of claim 1, wherein the mutans streptococcus strain is agenetically modified strain that is lactate dehydrogenase-deficient. 9.A food composition comprising: (a) (i one or more isolated Streptococcusoralis strains; (ii) or one or more isolated strains of Streptococcusuberis; (iii) or one or more isolated Streptococcus oralis strains andone or more isolated strains of Streptococcus uberis; and (b) one ormore isolated mutans streptococcus strains, wherein the mutansstreptococcus strains are lactate dehydrogenase-deficient.
 10. The foodcomposition of claim 9, wherein the mutans streptococcus strains areselected from the group consisting of S. rattus, S. cricetus, S. mutans,S. sobrinus, S. downeii, S. macacae, and S. ferus.
 11. The foodcomposition of claim 9, wherein the mutans streptococcus strain is anaturally-occurring mutant that is lactate dehydrogenase-deficient. 12.The food composition of claim 9, wherein the mutans streptococcus strainis a genetically modified strain that is lactatedehydrogenase-deficient.
 13. A dentifrice, oral rinse, chewing gum,lozenge, or topical agent composition comprising: (a) (i) one or moreisolated Streptococcus oralis strains; (ii) or one or more isolatedstrains of Streptococcus uberis; (iii) or one or more isolatedStreptococcus oralis strains and one or more isolated strains ofStreptococcus uberis; and (b) one or more isolated mutans streptococcusstrains, wherein the mutans streptococcus strains are lactatedehydrogenase-deficient.
 14. The composition of claim 13, wherein themutans streptococcus strains are selected from the group consisting ofS. rattus, S. cricetus, S. mutans, S. sobrinus, S. downeii, S. macacae,and S. ferus.
 15. The composition of claim 13, wherein the mutansstreptococcus strain is a naturally-occurring mutant that is lactatedehydrogenase-deficient.
 16. The food composition of claim 13, whereinthe mutans streptococcus strain is a genetically modified strain that islactate dehydrogenase-deficient.
 17. A method for maintaining oralhealth of a subject comprising administering the composition of claim 1to an oral cavity of a subject.
 18. The method of claim 17, wherein thesubject is a mammal.
 19. The method of claim 17, wherein the compositionis administered to the subject about once a day, about once a week, orabout once a month.
 20. The method of claim 17, wherein maintaining oralhealthy comprises the treatment, prevention or both treatment andprevention of periodontitis, dental caries, Candida or fungalovergrowth, halitosis, xerostomia-induced dental caries or periodontaldisease, oral bacterial infections, oral bacterial disease, oral woundsor a combination thereof.
 21. A method of non-persistently colonizing anoral cavity of a subject with therapeutically-effective bacteriacomprising administering to the oral cavity of the subject a combinationcomprising: (a) (i) one or more isolated Streptococcus oralis strains;(ii) or one or more isolated strains of Streptococcus uberis; (iii) orone or more isolated Streptococcus oralis strains and one or moreisolated strains of Streptococcus uberis; and (b) one or more isolatedmutans streptococcus strains, wherein the mutans streptococcus strainsare lactate dehydrogenase-deficient.
 22. The method of claim 21, whereinthe combination is administered to the subject about once a day, aboutonce a week, or about once a month.
 23. The method of claim 21, whereinthe subject is a mammal.